PROJECT SUMMARY/ABSTRACT Current anti-tuberculosis (TB) strategies require months of treatment, and the development of multi-drug re- sistant mutants of M. tuberculosis (Mtb) has increased both the complexity and cost of treatment. As such, the NIAID recently proposed targeting infected individuals? responses to infection as a means to enhance anti-TB defenses. These ?host-directed therapies? could be combined with proven antibiotic strategies against TB, providing an overall enhancement of treatment and patient care. Amino acids are integral to immune function, yet there is a fundamental gap in understanding the therapeutic potential of targeting amino acid metabolism during disease. The long-term goal is to define the interplay between amino acid metabolism and immune re- sponses, providing new therapeutic avenues to manipulate immune activity. The objective of this study is to identify the role of L-citrulline metabolism on macrophage (M?)-mediated immune responses to Mtb. The ap- plicant will use Mtb H37Rv infection in human and mouse M?s, as well as in vivo infection in mice, to examine L-citrulline metabolism during Mtb infection. The central hypothesis is that L-citrulline metabolism is required for anti-TB M? activity and can be harnessed to assist host defense to TB in vivo. The hypothesis is support- ed as a) L-citrulline enhances M? NO production and anti-Mtb activity in vitro, b) L-citrulline metabolism by myeloid cells is necessary for mycobacterial defenses in vivo, c) L-citrulline supplementation decreases lung mycobacterial burden, and d) lung M?s are the predominant L-citrulline utilizing cells during infection. The ra- tionale for the proposed research is that uncovering mechanics of immune-mediated infection control will likely lead to novel methods for treating those infected with Mtb ? which kills well over 1 million annually. The appli- cant will test the central hypothesis by investigating three specific aims: 1) to examine how L-citrulline is uti- lized in Mtb-infected M?s, 2) to define the metabolism of L-citrulline in human M?s infected with Mtb, and 3) to identify how harnessing L-citrulline metabolism enhances anti-mycobacterial host defenses. Under the first and second aims, the applicant will utilize a combination of innovative cell culture and mass spectrometry ap- proaches with titrating amino acid concentrations to determine the benefit(s) of L-citrulline metabolism in hu- man and mouse M?s. These experiments will define the mechanistic consequences of this pathway that will complement in vivo experiments under the third aim, where the applicant will use an original approach to en- hance Mtb clearance in the lungs by supplementing mice with L-citrulline. The proposed research is significant as we anticipate harnessing L-citrulline metabolism will augment M?-mediated control of TB, and in combina- tion with anti-mycobacterial antibiotic therapy will result in efficient treatment strategies for patients suffering with TB. This research is also expected to have broad implications on host defense mechanisms ? enhancing pathogen control and individual components of the immune system by altering amino acid metabolism.